Repeated sub-optimal photodynamic treatments with pheophorbide a induce an epithelial mesenchymal transition in prostate cancer cells via nitric oxide.

Photodynamic therapy (PDT) is a clinically approved treatment that causes a selective cytotoxic effect in cancer cells. In addition to the production of singlet oxygen and reactive oxygen species, PDT can induce the release of nitric oxide (NO) by up-regulating nitric oxide synthases (NOS). Since non-optimal PDT often causes tumor recurrence, understanding the molecular pathways involved in the photoprocess is a challenging task for scientists. The present study has examined the response of the PC3 human metastatic prostate cancer cell line following repeated low-dose pheophorbide a treatments, mimicking non-optimal PDT treatment. The analysis was focused on the NF-kB/YY1/RKIP circuitry as it is (i) dysregulated in cancer cells, (ii) modulated by NO and (iii) correlated with the epithelial to mesenchymal transition (EMT). We hypothesized that a repeated treatment of non-optimal PDT induces low levels of NO that lead to cell growth and EMT via the regulation of the above circuitry. The expressions of gene products involved in the circuitry and in EMT were analyzed by western blot. The findings demonstrate the cytoprotective role of NO following non-optimal PDT treatments that was corroborated by the use of L-NAME, an inhibitor of NOS.

The Role Of Nitric Oxide After Repeated Low Dose Photodynamic Treatments In Prostate Carcinoma Cells.

Photodynamic therapy (PDT) is a clinically approved treatment that causes a selective cytotoxic effect in cancer cells. In addition to the production of singlet oxygen and reactive oxygen species, PDT can induce the release of nitric oxide (NO) by up-regulating nitric oxide synthases (NOS). Since non-optimal PDT often causes tumor recurrence, understanding of the molecular pathways involved in the photoprocess is a challenging task for scientists. The present study has examined the response of the PC3 human metastatic prostate cancer cell line, following repeated low-dose pheophorbide a treatments, mimicking non-optimal PDT treatment. The analysis was focused on the NF-kB/YY1/RKIP circuitry as it is (i) dysregulated in cancer cells (ii) modulated by NO and (iii) correlated with the epithelial to mesenchymal transition (EMT). We hypothesized that a repeated treatment of non-optimal PDT induces low levels of NO that lead to cell growth and EMT via regulation of the above circuitry. The expressions of gene products involved in the circuitry and in EMT were analyzed by western blot. The findings demonstrate the cytoprotective role of NO following non-optimal PDT treatments that was corroborated by the use of l-NAME, an inhibitor of NOS.

Nitric oxide-mediated activity in anti-cancer photodynamic therapy.

Cell recurrence in cancer photodynamic therapy (PDT) is an important issue that is poorly understood. It is becoming clear that nitric oxide (NO) is a modulator of PDT. By acting on the NF-κB/Snail/RKIP survival/anti-apoptotic loop, NO can either stimulate or inhibit apoptosis. We found that pheophorbide a/PDT (Pba/PDT) induces the release of NO in B78-H1 murine amelanotic melanoma cells in a concentration-dependent manner. Low-dose PDT induces low NO levels by stimulating the anti-apoptotic nature of the above loop, whereas high-dose PDT stimulates high NO levels inhibiting the loop and activating apoptosis. When B78-H1 cells are treated with low-dose Pba/PDT and DETA/NO, an NO-donor, intracellular NO increases and cell growth is inhibited according to scratch-wound and clonogenic assays. Western blot analyses showed that the combined treatment reduces the expression of the anti-apoptotic NF-κB and Snail gene products and increases the expression of the pro-apoptotic RKIP gene product. The combined effect of Pba and DETA/NO was also tested in C57BL/6 mice bearing a syngeneic B78-H1 melanoma. We used pegylated Pba (mPEG-Pba) due to its better pharmacokinetics compared to free Pba. mPEG-Pba (30 mg/Kg) and DETA/NO (0.4 mg/Kg) were i.p. injected either as a single molecule or in combination. After photoactivation at 660 nM (fluence of 193 J/cm(2)), the combined treatment delays tumor growth more efficiently than each individual treatment (p<0.05). Taken together, our results showed that the efficacy of PDT is strengthened when the photosensitizer is used in combination with an NO donor.

Efficient silencing of bcr/abl oncogene by single- and double-stranded siRNAs targeted against b2a2 transcripts.

In this work, double- and single-stranded small-interference RNAs (siRNAs) were designed to knock down the bcr/abl oncogene in leukaemia KYO-1 cells. The siRNA molecules were targeted against two distinct sites encompassing the b2a2 junction of the bcr/abl transcripts. The siRNAs were able to reduce the levels of both bcr/abl mRNA and protein p210(BCR/ABL). Conversely, control siRNAs bearing 3 or 4 base-pair substitutions did not produce any inhibitory effect. The designed siRNAs were also found to be active in KCl22 cells, which harbor the b2a2 junction, but not in K562 cells, which, by contrast, harbor the b3a2 junction. The anti-b2a2 siRNAs promoted biological effects on KYO-1 cells, because the bcr/abl suppression resulted in the inhibition of cell growth and colony formation in agar and activation of apoptosis and upregulation of the cell-cycle inhibitor p27 protein. The bioactivity of the designed siRNAs is discussed in terms of internal stability of the RNA duplexes. Our data suggest that siRNAs can be considered strong tools for functional analysis of bcr/abl and for developing molecular therapeutic approaches to leukaemia.

Antigene effect in K562 cells of a PEG-conjugated triplex-forming oligonucleotide targeted to the bcr/abl oncogene.

Triplex-forming oligonucleotides are able to modulate gene expression by site-specific binding to genomic DNA. Their use as therapeutic agents is limited by inefficient cellular uptake, scarce nuclear internalization, and oligonucleotide self-aggregation. In this study, we demonstrate that a 13-mer AG motif oligonucleotide covalently linked to a high-molecular mass (9000 Da) polyethylene glycol (PEG ODN(13)) exhibits uptake and biological properties that are superior to those of the nonconjugated isosequence analogue (free ODN(13)). Band-shift and footprinting experiments showed that PEG ODN(13) forms a stable triple helix (apparent K(d) between 10(-6) and 10(-7) M in 50 mM Tris-acetate, 10 mM MgCl(2), pH 7.4, 37 degrees C) with a natural polypurine-polypyrimidine target located in the 5' flanking region of the human bcr/abl oncogene. Confocal laser microscopy performed on unfixed live cells stained with hexidium iodide as well as on glass-fixed cells stained with propidium iodide showed that fluorescein-labeled PEG ODN(13) is far more efficiently taken up and internalized in the nucleus by K562 and HeLa cells than the nonconjugated free ODN(13). It was found that PEG ODN(13) specifically downregulated the transcription of bcr/abl mRNA at 65 +/- 5% with respect to control and inhibited cell growth by 32 +/- 3% in a 3 day liquid culture assay. Moreover, PEG ODN(13) was more resistant against S1 and fetal bovine serum nucleases than free ODN(13), and less inclined to self-associate into multistrand structures in solution. Taken together, these results provide useful elements for designing artificial transcription repressors with enhanced potency in vivo.